In the following reply, we would like to address point-by-point the comments made by Banerjee et al. in response to our article entitled “Memantine Blocks α7* Nicotinic Acetylcholine Receptors More Potently than N-Methyl-d-aspartate Receptors in Rat Hippocampal Neurons” (J Pharmacol Exp Ther312:1195–1205). The disclosure of this information will be extremely helpful for a better understanding of the outcomes of the clinical trials with memantine in different stages of Alzheimer's disease (AD).

The pathophysiology of AD is very complex and involves alteration of the amyloid precursor protein (APP) processing, excessive deposits of τ aggregates, excitotoxicity, and significant alterations in numerous neurotransmitter systems in the brain. Although the cause of AD remains undetermined, one of the most significant neuropathological hallmarks of the disease is the early development of nicotinic cholinergic deficits in the brain. Based on this knowledge, treatments presently approved by the Food and Drug Administration (FDA) for patients with mild-to-moderate AD include the cholinesterase inhibitors donepezil and rivastigmine, as well as galantamine, a drug that acts as a weak cholinesterase inhibitor and a nicotinic allosteric potentiating ligand (Doody, 2003). The N-methyl-d-aspartate (NMDA) receptor antagonist memantine is the only FDA-approved treatment for patients with moderate-to-severe AD (Reisberg et al., 2003). The ability of memantine to improve cognition in these patients seems to result from a selective inhibition of the excessive tonic NMDA receptor activity that accounts for the neurodegeneration observed in AD.

In their comments, Banerjee et al. indicate that recent clinical studies have demonstrated beneficial effects of memantine in patients with mild-to-moderate AD. The clinical data from Peskind et al., which have been presented only in the format of research posters at the 17th Meeting of the American Association for Geriatric Psychiatry (Baltimore, MD; February 21–24, 2004) and the XXIV Collegium Internationale Neuro-Psychopharmacologicum Congress (Paris, France; June 20–24, 2004), has been recently reviewed in Areosa et al. (2004). Peskind et al. enrolled approximately 400 patients that had been diagnosed with mild-to-moderate AD and assigned these patients to treatment with memantine (20 mg/day) or placebo. At the end of the 24-week treatment, patients receiving memantine showed no significant improvements in the activities of daily living. Memantine caused an apparent small, albeit significant improvement in cognition, behavior, and mood, and the outcome of the clinician's interview-based impression of change-plus caregiver input also favored memantine. However, the Cochrane Database Systematic Review of “Memantine on Dementia” reports the existence of two finished but unpublished clinical trials in which memantine failed to improve cognitive or global measures in patients with mild-to-moderate AD. Thus, at this point, we feel that the effects of memantine on mild-to-moderate AD patients are unknown at best. Nevertheless, it has been reported that, based in part on results from Peskind et al., Forest Laboratories, Inc. intended to file a Supplemental New Drug Application with the FDA for approval of memantine for treatment of patients with mild-to-moderate AD (Rosack, 2004). These clinical findings and the presently available preclinical data on the inhibitory actions of memantine in the nicotinic cholinergic system in the brain (Buisson and Bertrand, 1998; Maskell et al., 2003; Aracava et al., 2005) suggest that the prospect of using this drug for treatment of mild-to-moderate AD should be examined with a great deal of awareness and caution.

Although the authors state in their comments that α7 nAChR expression is relatively unaffected in AD regardless of the disease, there are plentiful reports of region-specific losses of α7 nAChRs in the AD brain (Banerjee et al., 2000; Perry et al., 2000; Court et al., 2001; Nordberg, 2001). In fact, the loss of α7 nAChRs is largely pronounced in the hippocampus of AD patients and not as significant in the cerebral cortex (Hellström-Lindahl et al., 1999; Wevers et al., 1999). It is also noteworthy that the degree of losses of cholinergic neurons and nAChRs (particularly α7 and α4β2) correlate well with the magnitude of cognitive decline in AD patients as the disease progresses from mild to moderate (Francis et al., 1985; Perry et al., 2000; Nordberg, 2001). In patients with more advanced stages of AD, such a correlation between nAChR loss and cognitive impairment does not seem to exist (Sabbagh et al., 2001). These findings support the clinical reports that nicotinic cholinergic enhancing therapies are more effective in patients with mild-to-moderate AD than in patients with moderate-to-severe AD (see Doody, 2003).

Based on the observation that nicotine-induced facilitation of LTP induction in the CA1 field of hippocampal slices could be mimicked by a high concentration of MLA (100 nM), Fujii et al. (2000) suggested that the effect of nicotine on LTP resulted primarily from agonist-induced α7 nAChR desensitization. A subsequent study performed a quantitative comparative analysis of the effects of nicotine, choline, and α-BGT on induction of LTP in the CA1 field of the hippocampus (Mann and Greenfield, 2003). In agreement with the findings reported by Fujii et al. (2000), the nAChR agonists nicotine and choline facilitated induction of LTP (Mann and Greenfield, 2003). Of interest, however, the effect of 1 mM choline on LTP induction was smaller in magnitude than that of 10 mM choline. It is well documented that 1 mM choline is sufficient to fully desensitize α7 nAChRs whereas 10 mM choline is needed to cause nearly full activation of these receptors (see Mike et al., 2000 and references therein). Furthermore, Mann and Greenfield (2003) reported that the effect of α-BGT on LTP induction was significantly smaller than that of choline or nicotine. Thus, desensitization explains only in part the facilitation of LTP induction by α7 nAChR agonists; receptor activation is an important determinant of the effect that these drugs have on synaptic plasticity in the hippocampus (Mann and Greenfield, 2003). These findings support the notion that α7 nAChR inhibition underlies the cognitive impairment caused by α7 nAChR antagonists, whereas α7 nAChR activation accounts for the memory-enhancing properties of α7 nAChR agonists in mammals (see Levin, 2002 and references therein).

Equally important were studies designed to investigate the mechanisms by which nicotine and α7 nAChR-selective agonists protect neurons in primary and organotypic cultures against degeneration triggered by different insults (Carlson et al., 1998; Ferchmin et al., 2003; Gahring et al., 2003; see also Dajas-Bailador and Wonnacott, 2004 and references therein). It seems that agonist-induced α7 nAChR desensitization is a determinant of the ability that nicotine and α7 nAChR agonists have to reduce early excitotoxic damage of hippocampal neurons in vitro (Ferchmin et al., 2003). In contrast, agonist-induced α7 nAChR activation is unquestionably essential for the neuroprotective effect that nicotinic agonists have in delayed neurotoxicity (see references above). Ca2+ entry and mobilization following α7 nAChR activation in neurons is critical for activation of signaling cascades that can prevent delayed neurodegeneration (reviewed in Dajas-Bailador and Wonnacott, 2004), which resembles the slowly evolving neuronal death occurring in AD and other neurode-generative disorders (see Beal, 1996).

In their letter, the authors make reference to recent studies that have suggested that α7 nAChR activation may contribute to AD pathophysiology and neuropathology. Wang et al. (2003) have reported that the amyloid peptide Aβ1-42, via specific interactions with α7 nAChRs, induces phosphorylation of the τ protein. It should be noted, however, that in that study Aβ1-42-induced τ phosphorylation was blocked by concentrations of α-BGT and MLA that are 1,000-fold higher than those needed to block ion flux through α7 nAChRs and was insensitive to blockade by the nonselective nAChR antagonist mecamylamine (Wang et al., 2003). Thus, it is unlikely that τ phosphorylation resulted from the activation of α7 nAChRs by Aβ1-42. The work of Dineley et al. (2001, 2002) provided evidence that in the hippocampus of transgenic mice, high levels of Aβ1-42 lead to increased expression of α7 nAChRs and that Aβ1-42, acting most likely as an α7 nAChR agonist, causes chronic activation and subsequently down-regulation of the extracellular signal-regulated-protein kinase. These results led to the hypothesis that α7 nAChR-mediated alterations in extracellular signal-regulated-protein kinase activity could be one of the mechanisms underlying the disruption of synaptic functions and plasticity and, ultimately, the cognitive impairment observed in AD (Dineley et al., 2001, 2002). Unfortunately, these findings and hypotheses resulted from experiments carried out using transgenic mouse lines that express a mutant human presenilin-1 (PS-1), a mutant human APP, or both mutant human PS-1/APP. As such, it is unclear whether they can be used to interpret the neuropathology of AD, because these animal models do not reproduce the neuropathology of this catastrophic disease (Dodart et al., 2002; Higgins and Jacobsen, 2003). In particular, expression of α7 nAChRs is known to be increased in the hippocampi of the APP and PS-1/APP mice, whereas levels of these receptors are known to be decreased in the hippocampi of AD patients. In addition, one of the hallmarks of AD, the large loss of brain cholinergic neurons, is absent in the brain of these transgenic mice (Hernandez et al., 2001). Finally, the APP and PS-1/APP mice both lack the τ pathology associated with AD (Higgins and Jacobsen, 2003).

Although the possible effects of memantine on the eye-blink conditioning response in AD patients are hitherto unknown, it has been demonstrated that the nicotinic cholinergic system plays a central role in modulating this form of associative learning that is impaired in Alzheimer's disease (Woodruff-Pak, 2001). The classic eye-blink conditioning response is improved by nicotine and α7 nAChR agonists (Leon-S et al., 1997; Woodruff-Pak et al., 2000) and impaired by nAChR antagonists (Woodruff-Pak, 2003). Therefore, it is troublesome that a single 30-mg dose of memantine can cause impairment of this response in young health subjects (Schugens et al., 1997).

The clinical relevance of the finding that memantine reduces the accuracy and rate of response of rats in a fixed consecutive number task (Willmore et al., 2001) has been questioned by Banerjee et al. In general, a decreased performance in fixed consecutive number tasks is indicative of disruption of working memory (Sanger, 1992). However, as the authors pointed out in their comments, the reduction of accuracy by memantine cannot be unambiguously attributed to memory impairment, because at the doses that memantine reduced response accuracy, it also decreased response rates (Willmore et al., 2001). Nevertheless, memantine decreased task accuracy with ED50 values ranging from 4.2 to 12.0 mg/kg (Willmore et al., 2001); these doses are significantly lower than those used experimentally to ameliorate memory impairment induced by excessive glutamatergic activation in rats (Misztal et al., 1996) and those recommended clinically for AD treatment (Reisberg et al., 2003). Identifying the doses at which memantine selectively decreases response accuracy in this test would probably depend on increasing the complexity of the task (Willmore, 2003). A more recent study demonstrated that 10 mg/kg memantine impairs retention of a reference memory-based spatial learning task (Creeley et al., 2004). Therefore, it seems that memantine improves cognitive processing impaired by excessive glutamatergic activity and impairs cognition under conditions in which glutamatergic activity is not overtly increased.

In closing, we would like to emphasize that different research groups have consistently demonstrated that losses of cholinergic neurons and nAChRs, particularly α7 and α4β2, correlate well with the cognitive decline in AD at early stages. There is also plentiful evidence that α7 nAChR activation is neuroprotective and improves memory, whereas α7 nAChR inhibition impairs memory. Thus, it can be concluded that should inhibition of α7 nAChRs by memantine take place in the human brain, it would decrease the effectiveness of the drug in the treatment of patients with mild-to-moderate AD, although being of no great significance in patients with moderate-to-severe AD, when apparently the degree of loss of nAChRs no longer correlates with the cognitive decline of the patients (Sabbagh et al., 2001). Our findings strongly substantiate the need of further preclinical studies to address the benefits of using memantine for the treatment of patients with mild-to-moderate AD. As of today, the results of the clinical trials of memantine on these patients have certainly been rather disappointing. In the 24-week trials, memantine has either been ineffective or only slightly effective in improving the overall conditions of patients with mild-to-moderate AD (reviewed in Areosa et al., 2004).